1. Field of the Invention
The present invention relates to a method of producing a fraction containing a high concentration of milk-derived basic cystatin from a milk-derived basic protein fraction.
The present invention also relates to a method of producing a decomposition product of the resulting fraction containing a high concentration of milk-derived basic cystatin by decomposing said fraction containing a high concentration of milk-derived basic cystatin using a protease.
Further, the present invention relates to a drink, food product, medicine and feed to which the fraction containing a high concentration of milk-derived basic cystatin and/or the decomposition product of the fraction containing a high concentration of milk-derived basic cystatin milk-derived basic cystatin are admixed, and if necessary, calcium and/or vitamins are additionally admixed, for the prevention and treatment of various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease.
2. Description of the Related Art
In recent years, the number of people suffering from various bone diseases, such as bone fractures and lumbago has been on the rise because of the aging population. Bone formation and bone resorption continuously take place in bone tissue and are well balanced in early life, but the balance is lost with an increase in bone resorption with aging for various reasons. If this unbalance continues for a long period of time, the bone tissue becomes fragile, which results in various bone diseases such as osteoporosis, bone fractures and lumbago. It is believed that if this uncoupling leaning toward bone resorption can be impeded, various bone diseases can be prevented.
Conventional methods to prevent and treat various bone diseases include (1) dietary supplementation of calcium, (2) moderate exercise, (3) sunbathing, and (4) administration of medicines. For dietary supplementation, calcium salts such as calcium carbonate and calcium phosphate, natural calcium supplements such as bovine bone powders, eggshells and fish bone powders are used. For moderate exercise, moderate running or walking are highly recommended. However, even moderate exercise is difficult for people who are physically weak and, not to mention, almost impossible for elderly people who are confined to bed. The third method, sunbathing, is good for the supplementation of activated vitamin D3, but not sufficient by itself. For the last method, administration of medicines, 1xcex1-hydroxy vitamin D3, calcitonin preparations or the like are known to be effective to cure and treat osteoporosis. It is believed that bone joint diseases, such as rheumatism, and periodontal disease can be treated by suppressing bone resorption since they are ultimately associated with the bone resorption.
The present inventors intensively searched for a milk whey protein fraction having an osteoblast growth stimulating factor, a bone resorption suppressing factor and bone strengthening activity, in order to obtain a material which is effective for the prevention and treatment of various bone diseases, bone joint diseases and periodontal disease. Namely, the present inventors fractionated proteins in milk, in particular milk whey, in an attempt to obtain a fraction having a suppressing activity on osteoclastic bone resorption. As a result, the present inventors found that a bone strengthening activity in a protein-peptide mixture which was obtained by treating a water soluble fraction of whey proteins with a reverse osmotic membrane or electrodialysis to remove salts (Japanese Patent Application Laid-open No. H4-183371). Furthermore, the present inventors found that a fraction obtained by treating an aqueous solution of this protein-peptide mixture with ethanol, heat, salts or an ultrafiltration membrane has a bone strengthening activity (Japanese Patent Application Laid-open No. H5-176715, Japanese Patent Application Laid-open No. H5-320066). The present inventors also found that basic proteins present in milk in trace amounts have a collagen synthesis stimulating activity in osteoblasts and bone resorption preventing activity (Japanese Patent Application Laid-open No. H7-207509).
Cystatin, a cysteine protease inhibitor, is a substance which inhibits proteolytic activity of cysteine proteases having an SH group in the active center and is found in animal tissues, cells, blood and urine. Further, a virus growth inhibiting activity is recognized as a useful activity of cystatin (Biochem. Biophys. Res. Commun., Vol. 127, p. 1072, 1985).
In recent years, the number of people suffering from osteoporosis caused by osteoclastic bone resorption has been rapidly increasing. Currently, a calcitonin drug is known as a medicine to suppress this osteoclastic bone resorption. However, a calcitonin drug is a hormonal agent used as a medicine, and the safety of its use as a food material has not been examined to this day. Further, production of calcitonin in bulk from animal tissues, cells, blood or urine for use as a food material has not been attained.
The present inventors tried to isolate and purify an active substance having a bone resorption suppressing activity from a basic protein fraction having a bone resorption suppressing activity, identified the resulting isolated and purified substance, and confirmed that this substance is a milk basic cystatin. Furthermore, the present inventors found that the milk basic cystatin has a suppressing activity more specifically on osteoclastic bone resorption, as compared with other kinds or types of cystatins from origins other than milk (Japanese Patent Laid-open No. 2000-281587, published Oct. 10, 2000, which is after the priority date of this application).
Under these circumstances, a method of producing a milk-derived basic cystatin usable as a food material in bulk and at a low cost is in need. Accordingly, an object of the present invention is to provide a method of producing a fraction containing a high concentration of basic cystatin from a milk-derived basic protein fraction.
Further, an object of the present invention is to provide a method of producing a decomposition product of the fraction containing a high concentration of milk basic cystatin since a protease decomposition product of the fraction containing a high concentration of milk basic cystatin is also known to have a bone resorption suppressing activity.
Furthermore, another object of the present invention is to provide a drink, food product, medicine or feed to which the fraction containing a high concentration of milk-derived basic cystatin and/or the decomposition product of the fraction containing a high concentration of milk-derived basic cystatin are admixed for the prevention and treatment of various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease.
The process for extracting basic cystatin from milk is broadly divided into (1) a process to extract a basic protein fraction from milk, and (2) a process to extract basic cystatin from the basic protein fraction. As explained below, a basic protein fraction can be extracted in great volume from milk volume by, for example, contacting milk with a cation exchange resin, and then eluting a fraction adsorbed on the resin with an elute having a salt concentration of 0.1-1.0 M. However, a further treatment is required for recovering basic cystatin at a high concentration such as 1% or higher by dry weight. Separating basic cystatin from other basic proteins can be accomplished to a certain degree by a method utilizing its physico-chemical characteristics, such as steps of heating a solution at 80xc2x0 or higher, recovering a supernatant by removing the resultant precipitate, adding alcohol to the supernatant, and removing the resultant precipitate. However, it is difficult to obtain basic cystatin at a high concentration by the above method. The present invention provides a method of extracting and recovering basic cystatin at a high concentration from a basic protein fraction. Namely, the present invention is a production method comprising the steps of: (1) preparing a composition containing a milk-derived basic protein fraction; (2) contacting said composition with a carrier containing a sulfuric acid group; (3) recovering a fraction which is not adsorbed onto the carrier; (4) subjecting said fraction to ultrafiltration using a membrane having a cut-off molecular weight of 10-50 kDa; and (5) recovering a permeate from said ultrafiltration membrane to recover a fraction containing milk basic cystatin from said permeate, or a production method comprising the steps of: (1) preparing a milk-derived basic protein composition; (2) contacting said milk-derived basic protein composition with an anion exchange resin; (3) recovering a fraction which is not adsorbed onto said resin; (4) contacting said fraction with a cation exchange resin; and (5) eluting and recovering a fraction which is adsorbed onto said resin with an eluent to recover a fraction containing milk basic cystatin. According to the present invention, a composition of a fraction containing milk basic cystatin containing approximately 1% or higher milk basic cystatin by dry weight can be produced in great volume (preferably, desalting treatment is conducted). In the above, preferably, the obtained fraction is subjected to desalting and concentration treatment such as ultrafiltration (a cut-off molecular weight of 10-50 kDa) or diafiltration.
For purposes of summarizing the invention and the advantages achieved over the prior art, certain objects and advantages of the invention have been described above. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.
Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow.
According to an embodiment of the present invention, a method of producing a fraction containing a high concentration of milk basic cystatin is characterized in that a milk-derived basic protein fraction is heated, if necessary, at 70-90xc2x0 C. (preferably at 80xc2x0 C. or higher), the resulting precipitate is removed to recover the supernatant, alcohol is added to this supernatant, at 30-80% preferably at a concentration of 70%), a fraction containing a high concentration of milk basic cystatin is recovered by removing the resulting precipitate. The above processes are not indispensable but useful because the concentration of basic cystatin can be raised to a certain degree. The recovered fraction is then brought into contact with the fraction with a carrier containing a sulfuric acid group (preferably by passing through a column a solution prepared by dissolving the basic protein fraction in a buffer solution having a pH of 4-8 at a final concentration of 1-5% by weight) to recover a fraction which is not adsorbed onto the carrier, and this fraction is then treated with an ultrafiltration membrane having a cut-off molecular weight of 10-50 kDa to recover the permeate. As a carrier containing a sulfuric acid group, a heparin-immobilized column and others.
Further, according to the present invention, a method of producing a decomposition product of the fraction containing a high concentration of milk basic cystatin is characterized in that the fraction containing a high concentration of milk basic cystatin produced by the abovementioned method is further decomposed with a protease.
Further, the present invention is a drink, food product, medicine or feed to which the fraction containing a high concentration of milk-derived basic cystatin and/or the decomposition product of the fraction containing a high concentration of milk-derived basic cystatin are admixed, and if necessary, calcium and/or vitamins are additionally added, for the prevention and treatment of various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease. The effective dose of a fraction containing milk basic cystatin or its decomposition product may be approximately 1 xcexcg-100 mg/100 g.
Basic cystatin can also be recovered at a high concentration from a fraction containing milk-derived basic protein by the following method. Namely, according to another embodiment of the present invention, a method of producing a fraction containing a high concentration of milk basic cystatin is characterized in that a milk-derived basic protein composition (preferably, a solution prepared by dissolving a basic protein in a buffer solution having a pH of 4-8 at a final concentration of 1-5% by weight) is brought into contact with an anion exchange resin to recover a fraction which is not adsorbed onto the resin, after which this fraction is brought into contact with a cation exchange resin to recover the target fraction by eluting a fraction which is adsorbed onto the resin with an eluent. As with the previously mentioned method, the recovered fraction is preferably subjected to desalting and concentration treatment.
Milk or a milk product, such as reconstituted milk, skimmed milk, and whey, can be used to obtain a milk-derived basic protein composition, a starting material of the present invention. The milk-derived basic protein composition which is present in milk only in trace amounts comprises primarily lactoferrin and lactoperoxidase. Accordingly the milk-derived basic protein composition can be obtained in bulk by bringing milk into contact with a cation exchange resin, and then eluting a fraction which is adsorbed onto the resin with an eluent having a salt concentration of 0.1-1.0 M.
The eluent to elute the fraction which is adsorbed onto the cation exchange resin can be a solution containing a salt such as 0.1-1.0 M sodium chloride.
Examples of resins to be used in the present invention include commercial Q Sepharose (Pharmacia) as an anion exchange resin and commercial S-Sepharose (Pharmacia) as a cation exchange resin.
The present invention also includes a method of producing a decomposition product of a fraction containing a high concentration of milk basic cystatin by decomposing the fraction containing a high concentration of milk basic cystatin produced by the abovementioned method, with a protease.
Further, the present invention includes a drink, food product, medicine or feed to which the fraction containing a high concentration of milk-derived basic cystatin and/or the decomposition product of the fraction containing a high concentration of milk-derived basic cystatin are admixed, and if necessary, calcium and/or vitamins are additionally added, for the prevention and treatment of various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease. In the above case (a method using an anion exchange resin and a cation exchange resin in combination), the effective dose of a fraction containing milk basic cystatin or its decomposition product may be approximately 5 xcexcg-500 mg/100 g, which is slightly higher than in the case of a method using a carrier containing a sulfuric acid group. This is because the purity of basic cystatin in the former is slightly lower than in the latter.
In addition, a method using an anion exchange resin and a cation exchange resin in combination, and a method using a carrier containing a sulfuric acid group can be combined to extract and recover basic cystatin. That is, by conducting one of the methods after conducting the other method, basic cystatin containing can be obtained at a high concentration.
According to the present invention, a safe fraction containing a high concentration of milk basic cystatin can be produced in bulk. Examples of milk to be used include raw milk, powdered milk, powdered skim milk, and reconstituted milk.
Further, a decomposition product of the fraction containing a high concentration of milk basic cystatin of the present invention is a peptide mixture prepared by restrictively decomposing the abovementioned fraction containing a high concentration of milk basic cystatin with a protease, such as trypsin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. In general, decomposition products exhibit higher activity than the fraction itself. The degree of decomposition of the fraction with an enzyme can be determined with reference to its bone resorption activity.
Further, in the present invention, the abovementioned fraction containing a high concentration of milk basic cystatin and/or decomposition product of the fraction containing a high concentration of milk basic cystatin are added to a drink, food product, medicine or feed. Namely, they can be added to a drink or food product such as milk, milk drinks, juices, jellies, biscuits, breads, noodles, and sausages, made into a form of tablet, powder, tooth paste, or mouthwash for medicinal use, or added to feed.
Further, a highly absorbable calcium is preferably admixed into the drink, food product, medicine and feed of the present invention for the prevention and treatment of various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease. Examples of the highly absorbable calcium include calcium chloride, calcium carbonate, calcium lactate, eggshells and milk-derived calcium. Vitamins effective for bone formation, such as vitamin D and vitamin K, can also be preferably admixed. A synergistic effect on the bone formation can be attained since these vitamins are different from milk basic cystatin in their actions. Further, milk basic cystatin is an excellent material for food processing because of its high heat stability.
In the present invention, a fraction containing a high concentration of milk basic cystatin and/or decomposition product of the fraction containing a high concentration of milk basic cystatin can be administered in an amount of about 1 xcexcg to 500 mg (preferably 5 xcexcg to 100 mg) per day for an adult in divided doses. In this way, various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease can be prevented and treated.
The present invention will be explained in detail in the following examples.